低温菌启动子分析及异源蛋白高效表达-微生物学报.PDFVIP

生 物 工 程 学 报 Chin J Biotech 2008, March 25; 24(3): 415-422 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ © 2008 Institute of Microbiology, CAS CSM, All rights reserved 研究报告 魏云林, 季秀玲, 林连兵, 井申荣 , , 650224 : 在已构建的能在低温菌和E. coli 中正常复制的启动子探针质粒的基础上, 筛选到了强启动子, 通过RT-PCR 评 估了启动子活性, 并通过引物延伸实验确定了转录起始位点和启动子核心序列。利用其中最强的启动子构建了低温蛋白 表达质粒, 使一个热不稳定α-淀粉酶在低温下(7℃)得到了高效表达, 表 达量达胞外总蛋白的35% 。显示出该表达系统具 有高效表达热不稳定蛋白质的潜在应用价值。 : 启动子, 表达系统, 热不稳定蛋白质 The Analysis of Promoters and the Effective Expression of Exogenous Proteins in a Cold-adapted Bacterium Yunlin Wei, Xiuling Ji, Lianbing Lin, and Shenrong Jing Biotechnology Research Center of Kunming University of Science and Technology, Kunming 650224, China Abstract: Based on the constructed promoter probe vectors that could replicate both in E. coli and in a cold-adapted bacterium, several candidate promoters were isolated and their activities were evaluated by RT-PCR. The transcription initiation sites and core sequence of promoters were determined by primer extension analysis. A low-temperature expression vector was constructed by using the strongest promoter and a thermolabile α-amylase gene was successfully overproduced under control of this promoter at low tem- perature (7℃), while the secreted α-amylase amounted up to 35% of the total extracellular proteins. The expression system is ex- pected to be useful for the production of thermolabile exogenous proteins at low temperatures. Keywords: promoter, expression system, thermolabile protein 80% [4] [1,2] , ,